Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches

Ubiquitination, a pivotal posttranslational modification of proteins, plays a fundamental role in regulating protein stability. The dysregulation of ubiquitinating and deubiquitinating enzymes is a common feature in various cancers, underscoring the imperative to investigate ubiquitin ligases and deubiquitinases (DUBs) for insights into oncogenic processes and the development of therapeutic interventions. In this review, we discuss the contributions of the ubiquitin–proteasome system (UPS) in all hallmarks of cancer and progress in drug discovery. We delve into the multiple functions of the UPS in oncology, including its regulation of multiple cancer-associated pathways, its role in metabolic reprogramming, its engagement with tumor immune responses, its function in phenotypic plasticity and polymorphic microbiomes, and other essential cellular functions. Furthermore, we provide a comprehensive overview of novel anticancer strategies that leverage the UPS, including the development and application of proteolysis targeting chimeras (PROTACs) and molecular glues.


Introduction
Ubiquitin (Ub) is composed of 76 amino acids and is found in all eukaryotic tissues [1].Ubiquitination is the second most common posttranslational modification of proteins following phosphorylation [2].Ubiquitination is a highly specific process of ATP-dependent cascade labeling substrate proteins with ubiquitin [3].Moreover, ubiquitin and its degradation by the proteasome constitute the ubiquitin-proteasome system (UPS), which is responsible for 80-90% of cellular proteolysis and 10-20% of autophagy [4].The ubiquitination modification involves a series of reactions mediated by a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3) (Fig. 1) [5].Ubiquitination plays a crucial regulatory role in the modulation of tumors, impacting cellular survival, proliferation, and differentiation.At the same time, ubiquitination is reversible, and ubiquitin or ubiquitin chains linked to substrate proteins can be removed by deubiquitinases (DUBs).
The emerging functions of ubiquitination and deubiquitination in regulating cancer hallmarks, including "evading growth suppressors, " "reprogramming energy metabolism, " "unlocking phenotypic plasticity, " "polymorphic microbiomes, " and "senescent cells, " have been revealed [6,7].The UPS can regulate the protein levels of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) in the tumor microenvironment (TME) and enhance the effectiveness of immunotherapy [8].For example, ubiquitin-specific protease 2 (USP2), a DUB, can stabilize PD-1 and promote tumor immune escape through deubiquitination [9].Moreover, the UPS also regulates tumor metabolic reprogramming.Recent investigations have revealed that the E3 ligase Parkin can facilitate the ubiquitination of pyruvate kinase M2 (PKM2) [10].In addition, the DUB OTU domain-containing ubiquitin aldehyde-binding protein 2 (OTUB2) interacts with PKM2 to inhibit PKM2 ubiquitination by the E3 ligase Parkin, enhancing glycolysis and accelerating colorectal cancer progression [11].Many new reports have recently revealed novel ubiquitination methods for cancer treatment, such as PROTACs and molecular glues.PROTAC technology is a valuable platform for driving the degradation of target proteins.ARV-110 (alias bavdegalutamide) and ARV-471 (alias vepdegestrant) represent the forefront of PROTAC drug development in clinical trials and have progressed to phase II trials [12].Compared to PROTACs, molecular glues have smaller molecular dimensions, simplifying the optimization of their chemical characteristics.A few molecular glue degradants have been identified.Notably, CC-90009 facilitates the ubiquitination-mediated degradation of G1-to-S phase transition 1 (GSPT1) by recruiting the E3 ligase complex CUL4-DDB1-CRBN-RBX1 (CRL4 CRBN ).It is in phase II clinical trials for leukemia therapy [13].ARV-110 is designed to selectively target and bind to the androgen receptor (AR), facilitating its degradation by recruiting an E3 ubiquitin ligase.Early data from the first-in-human phase I study revealed the safety and tolerability of ARV-110 in patients diagnosed with metastatic castration-resistant prostate cancer [14].Our research team recently identified a drug that promotes protein ubiquitination and degradation.Indomethacin, for instance, diminishes the growth and recurrence of esophageal squamous cell carcinoma (ESCC) by enhancing the E3 ligase synovial apoptosis inhibitor 1 (SYVN1)mediated ubiquitination of integrin αv (ITGAV) [15].We also discovered that honokiol directly interacts with keratin 18 (KRT18), inhibiting melanoma growth by inducing KRT18 ubiquitination and degradation [16].
Fig. 1 The processes of ubiquitination and deubiquitination occur within the ubiquitin-proteasome system (UPS).Ubiquitination involves the sequential action of three enzyme classes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3).Initially, E1 enzymes activate ubiquitin (Ub) in an ATP-dependent process.Subsequently, the activated Ub is transferred to E2 enzymes via a thioester bond.The final step is catalyzed by E3 ligases, which facilitate the transfer of Ub from E2 to the target substrate protein, marking it for degradation In this review, we integrate the ubiquitination and deubiquitination processes with the 14 hallmarks of cancer.We clarify the fundamental mechanisms and functions of ubiquitination and deubiquitination in tumor suppression, metabolic reprogramming, immune evasion, phenotypic plasticity, polymorphic microbiomes, and other essential cellular functions, focusing on recent developments.Finally, we explore the therapeutic potential of targeting the UPS in cancer therapy.

Functions of various types of ubiquitination in cancer
Ubiquitination can be categorized into monoubiquitination, multimonoubiquitination, homotypic polyubiquitination, and heterotypic polyubiquitination (Fig. 2) [4].Monoubiquitination refers to the attachment of a single ubiquitin protein to a substrate protein.When multiple ubiquitin proteins are attached to different lysine residues on the same substrate protein, it is termed multimonoubiquitination. Ubiquitin itself contains eight potential linkage sites, which include seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) and one N-terminal methionine residue (M1) [17].These sites allow for the formation of polyubiquitin chains through further ubiquitin attachment.Homotypic polyubiquitination occurs when ubiquitin proteins are linked through the same residue type, creating a uniform chain [4].In contrast, heterotypic polyubiquitination involves ubiquitin proteins linked through different residue types, resulting in mixed or branched chains [18].When more than one ubiquitin molecule is simultaneously linked to a single ubiquitin molecule, the resulting ubiquitin chain is referred to as a branched ubiquitin chain [18].A family of proteins, known as ubiquitin-like proteins (UBLs), shares structural and functional characteristics with ubiquitin, playing similar roles in modulating protein activity and cellular processes.This family encompasses proteins such as neural precursor cell-expressed developmentally downregulated 8 (NEDD8), small ubiquitinrelated modifier (SUMO), and interferon-stimulated gene 15 (ISG15) [17].A ubiquitin-like modified chain means that the substrate or ubiquitin is modified by a ubiquitin-like protein.In addition, ubiquitin can also be posttranslationally modified through phosphorylation and acetylation, which is called a chemically modified ubiquitin chain.The formation of mixed ubiquitin chains, Fig. 2 The various types of ubiquitin (Ub) linkages are as follows.a Mono-ubiquitination: A single ubiquitin protein is attached to a substrate protein.b Multi-monoubiquitination: Multiple ubiquitin proteins are each linked to different sites on the same substrate protein.c Homotypic polyubiquitination: Ubiquitin can bind to another ubiquitin through one of its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) or the N-terminal methionine residue (M1).Multiple identical ubiquitin proteins form a chain, which is then attached to a substrate protein.d Linear ubiquitination: A specific form of homotypic polyubiquitination where ubiquitin molecules are connected via Met1 linkages.e Mixed ubiquitin chain: A ubiquitin can be linked by two or more different connection methods within the same polymerization reaction, resulting in mixed ubiquitin chains.f Branched ubiquitin chain: Ubiquitin proteins in a chain are modified by adding more ubiquitin proteins at different binding sites.g Ubiquitin-like modified chain: A ubiquitin protein in the chain is linked to a ubiquitin-like protein.h Chemically modified chain: Ubiquitin proteins in the chain are modified by other protein modifications, such as phosphorylation or acetylation.The formation of mixed ubiquitin chains, branched ubiquitin chains, ubiquitin-like modified chains, and chemically modified ubiquitin chains are collectively referred to as heterotypic polyubiquitination branched ubiquitin chains, ubiquitin-like modified chains, and chemically modified ubiquitin chains are collectively referred to as heterotypic polyubiquitination [4].

Monoubiquitination
Previous studies have shown that monoubiquitination of proteins acts as a signal for DNA repair, signal transduction, and phagocytosis in vivo [19,20].Recently, histone monoubiquitination has been widely studied.Histone monoubiquitination often occurs on H2A and H2B.Ring finger protein 2 (RNF2) is an E3 ligase with a RING domain [21].RNF2 facilitates the monoubiquitination of histone H2A at lysine 119, leading to the recruitment of E-cadherin to the promoter region and subsequent transcriptional repression of E-cadherin.This mechanism contributes to enhancing the metastatic potential of hepatocellular carcinoma [22].In addition, ubiquitinconjugating enzyme E2T (UBE2T) regulates the monoubiquitination of the histone variant H2AX (γH2AX).This process induces the phosphorylation of cell cycle checkpoint kinase 1 (CHK1), thereby enhancing the radioresistance of hepatocellular carcinoma cells [23].Monoubiquitination also plays an essential role in immune escape.Metastasis suppressor protein 1 (MTSS1) promotes the monoubiquitination of the immune checkpoint PD-L1 at K263 mediated by the E3 ligase atrophin-interacting protein 4 (AIP4), which leads to the internalization of PD-L1, endosomal transport, and lysosomal degradation, thus inhibiting the immune escape of lung adenocarcinoma [24].Additionally, the ubiquitin-binding enzyme E2B (UBE2B) can facilitate the monoubiquitination of the transcription regulator zinc finger MYM-type protein 2 (ZMYM2) mediated by the ubiquitin E3 ligase ring finger protein 73 (RNF73), thereby promoting the growth of ovarian cancer [25].These observations suggest that the monoubiquitination of proteins primarily affects the growth, metastasis, radiation resistance, and immune escape of cancer cells by affecting DNA repair and gene transcription.

Linear ubiquitination
The ubiquitin chains assembled by M1 are called linear ubiquitin chains.These chains are assembled exclusively by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and are disassembled by OTU deubiquitinase with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) [26,27].LUBAC consists of HOIL-1 interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), and SHANK-associated RH domain-interacting protein (SHARPIN) [28].The impact of linear ubiquitin chains on cancer has been extensively investigated.Met1-Ub signaling plays a vital role in many aspects of cancer through NF-κB regulation.HOIP promotes lymphoma by activating NF-κB signal transduction, indicating that LUBAC is a viable therapeutic target for B-cell lymphoma [29].Epsin, a member of the ubiquitin-binding endocytosis adaptor protein family, engages with the linear ubiquitin chain assembly complex (LUBAC) to facilitate the linear ubiquitination of the NF-κB essential modulator (NEMO).This interaction is implicated in the progression of breast cancer [30].Protein kinase transforming growth factor β-activated kinase 1 (TAK1) is the main mediator of NF-κB activation in the LUBAC-dependent mechanism.Targeting LUBAC or TAK1 may be an attractive therapeutic strategy for A20-mutant Hodgkin's lymphoma [31].RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) was first identified as an essential component of LUBAC and promoted NF-κB signal transduction during the immune response [32].Furthermore, the phosphorylation of OTULIN facilitates the activation of the genotoxic Wnt/ β-catenin pathway, thereby augmenting drug resistance in breast cancer [28].Consequently, the Met1-linked linear ubiquitin chain acts as an essential positive modulator of NF-κB signaling pathways, playing pivotal roles in oncogenesis, inflammation, and immune regulation.

K48-linked polyubiquitination
K48-linked polyubiquitination is the most widely studied type and the main connection type in cells.It mainly marks proteins that are recognized and degraded by the 26S proteasome and targets proteins for proteasomal degradation [18].The E3 ligase tripartite motif protein 7 (TRIM7) can directly interact with the tyrosine kinase Src, induce the ubiquitination of Lys48-linked Src, reduce the abundance of the Src protein in hepatocellular carcinoma cells, and inhibit the progression of hepatocellular carcinoma [33].Recently, a new circRNA involved in hypoxia reactions named circular insulin-induced gene 1 (circINSIG1) was identified.CircINSIG1 encodes the protein circINSIG1-121, which has 121 amino acids.By recruiting the E3 ligase CUL5-ASB6 complex, circIN-SIG1-121 promotes the ubiquitination of the critical cholesterol metabolism regulator INSIG1 at the K48 linkage of lysine 156 and lysine 158, thus inducing cholesterol biosynthesis and promoting colorectal cancer proliferation and metastasis [34].In addition, the E3 ligase MG53 catalyzes the K48-linked ubiquitination and subsequent degradation of cyclin D1, thus inhibiting the growth of colorectal cancer [35].However, in the ubiquitinating enzyme family, studies have shown that methyltransferase 5, N6-adenosine (METTL5) regulates the translation of USP5 and suppresses K48-linked ubiquitination of c-Myc, thus reprogramming glucose metabolism and promoting the progression of hepatocellular carcinoma [36].Therefore, as the most widely studied ubiquitination form, K48-linked polyubiquitination plays a key role in various aspects of cancer by promoting the degradation of corresponding proteins.

K63-linked polyubiquitination
K63-linked polyubiquitination participates in signal assembly and promotes the autophagic degradation of protein substrates.It can also regulate nondegradative processes, such as protein transport, DNA repair, and protein kinase activation [37].The AB22A-NeoF1 fusion gene encodes the Rab22a-NeoF1 fusion protein, which coordinates various mechanisms to facilitate lung metastasis in osteosarcoma [38].The E3 ligase STIP1 homology and U-box-containing protein 1 (STUB1) catalyzes the K63-linked ubiquitination of K112 of the Rab22a-NeoF1 fusion protein, which promotes the lung metastasis of osteosarcoma [39].K63-linked polyubiquitination also plays an important role in immune escape.For instance, the E3 ligase TRIM28 promotes the K63linked ubiquitination of TANK-binding kinase 1 (TBK1).It activates the TBK1-IRF1 and TBK1-mTOR pathways, thus enhancing the transcription of PD-L1 and promoting the escape of gastric cancer cells from immune surveillance [40].In addition, mind bomb homolog 2 (MIB2) catalyzes the ubiquitination of PD-L1 at the K63 linkage, but not its degradation, and promotes tumor immune escape [41].In addition, anillin (ANLN) is a mitotic protein that can promote the formation of contractile rings and cell division.The results showed that USP10 removes the K11-and K63-linked ubiquitin chains of ANLN through its ubiquitinating enzyme activity and prevents the ubiquitin-mediated degradation of ANLN, effectively inhibiting the cell cycle procession of ESCC [42].Taken together, K63-linked polyubiquitination plays an important role in cancer metastasis, immune escape, and the cell cycle.

Other types of polyubiquitination
Relatively few modified substrates and functions of "atypical" ubiquitin chains (K6, K11, K27, K29, K33, and M1 chains) are known [43,44].The ubiquitination of K11 is mainly related to UBE2S.Previous research revealed that UBE2S stabilizes β-catenin via K11-linked ubiquitination, contributing to the development of colorectal cancer [45].In addition, UBE2S interacts with TRIM21, which degrades lipoma preferred partner (LPP) through ubiquitination linked with K11 and promotes the lymphatic metastasis of bladder cancer [46].The K29-linked ubiquitin chain plays a significant role in driving cancer invasion and metastasis and in the positive regulation of immunity [47].Recent studies have demonstrated that ring finger protein 167 (RNF167) activates mTORC1 and promotes the occurrence of breast cancer by targeting and degrading K29-linked ubiquitinated cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1).In addition, this observation confirmed that RNF167 is a therapeutic target of breast cancer [48].
In addition to eight homotypic polyubiquitination modifications, heterotypic polyubiquitination modifications also occur widely in cells [18].These modifications predominantly involve mixed and branched polyubiquitination, characterized by the formation of polyubiquitin chains on substrates that feature two distinct types of lysine linkages, resulting in complex ubiquitin chain configurations [49].Poly(A)-binding protein, cytoplasmic 1 (PABPC1), is an extensively studied protein, and recent research has revealed its involvement in the tumorigenesis of numerous cancers.CDC2-like kinase 2 (CLK2) is a bispecific kinase, that facilitates the phosphorylation of diverse proteins, and an increasing amount of data indicate that CLK2 functions as an oncogenic kinase [50].USP10 can reverse K27/29-linked ubiquitination of PABPC1 and upregulate the translation of CLK2, thus promoting tumor development of pancreatic ductal adenocarcinoma (PDAC) [51].

Sustained proliferative signaling
The ability to maintain cancer cell proliferation is a fundamental characteristic of cancer cells.Normal cells can control the production and release of growth-promoting signals.However, cancer cells can escape the control of these signals and obtain sustained proliferative stimulation (Fig. 3) [6].

Epidermal growth factor receptor (EGFR) signaling pathway
The EGFR-dependent signaling pathway maintains cell proliferation, and its dysregulation increases cancer cell proliferation [52,53].EGFRvIII is the most common mutation in gliomas [54].Tumors with EGFRvIII mutations exhibit an increase in phosphorylation at Y371 of casitas B-lineage lymphoma (Cbl), a critical residue regulating E3 activity.The conformational changes in Cbl are essential for EGFR ubiquitination in vitro.EGFRvIII mutations can reduce EGFR protein degradation in tumors by inhibiting Cbl activation (Table 1) [55,56].It was indicated that β-Element, a traditional Chinese herb, played an anti-tumor and anti-metastatic role in multidrug-resistant (MDR) gastric cancer by suppressing EGFR levels through Cbl-b upregulation [57].Additionally, a high expression level of Cbl-b is significantly associated with improved prognosis in patients with lung adenocarcinoma, suggesting its potential as a prognostic biomarker for better clinical outcomes [58].Recent studies have reported that the overexpression of DUBs such as Cezanne-1, USP22, and USP25 in cancer cells prevents EGFR degradation [59][60][61].In clinical studies, Cezanne-1 is often amplified in tumor samples from breast cancer patients, with elevated levels of its mRNA associated with a poor prognosis [62].Similarly, there is a significant association between USP22 levels and poor prognosis in neuroblastoma [63].Notably, an in vivo study suggested that USP25/28 inhibitor showed a potent anti-tumor effect on pancreatic cell-derived xenograft (CDX) mouse model, implying combining DUB inhibitors with EGFR inhibitors or chemotherapeutic agents may enhance therapeutic efficacy [64].In conclusion, these observations suggest that E3 ligases and DUBs regulate EGFR signaling, opening new avenues for targeted therapeutic strategies against cancer cell proliferation.

MAPK signaling pathway
The MAPK pathway includes the RAS-RAF-MEK-ERK pathway and the JNK and p38 pathways [215].In the ERK1/2 signaling pathway, E3 ligases RNF149 and FBXW7 regulate the stability of B-Raf in colon adenocarcinoma (COAD), leading to its degradation.This degradation inhibits ERK1/2 signaling and tumor cell growth [216,217].Additionally, the deubiquitinating enzyme USP10 protects C-Raf from degradation in ectopic endometrial stromal cells [218].The UPS also regulates the ERK signaling pathway by affecting MEK1/2 expression, with USP21 involved in maintaining MEK2 stability and activating ERK signaling in hepatocellular carcinoma.The high expression of USP21 in hepatocellular carcinoma is associated with a lower survival rate among hepatocellular carcinoma patients.The research identified new clinical treatment strategies targeting the USP21-MEK2 interaction and its functions [91].Activation of the MAPK pathway is known to promote the progression of hepatocellular carcinoma [219].Targeted therapies against the MAPK pathway have become a focal point, with inhibitors targeting this pathway currently undergoing clinical trials [220,221].Deubiquitinating enzymes like USP2A, USP8, and USP15 have been identified as crucial modulators that promote MAPK pathway molecules deubiquitination [222].Interestingly,   USP8 knockdown can overcome gefitinib and erlotinib resistance [223,224].However, only a few USP8 inhibitors have been identified.Tian et al. discovered that DC-U4106 effectively binds to USP8 with a KD value of 4.7 μM and significantly suppresses breast cancer tumor growth while exhibiting minimal toxicity in a xenograft model [225].The JNK1/2/3 and p38 signaling cascades involve multiple MAPKKKs and MAPKKs, including MEKK1/2/3/4, TAK1, and ASK1, which can be activated by various stimuli [226].USP4 stabilizes the TAK1 protein level in ESCC cells through deubiquitination [82].USP4/TAK1 plays a critical role in the progression of esophageal squamous cell carcinoma (ESCC) by regulating proliferation, migration, and invasion.Silencing USP4 has been shown to inhibit tumor proliferation in ESCC nude mouse models.Moreover, the USP4 inhibitor, Neutral Red, can suppress ESCC progression both in vitro and in vivo [82].Another UDB molecule, USP15, can also target TAK1 and inhibit the proteasomal degradation of TAK1-binding protein 2 (TA B2) [227].Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK that initiates cell death and inflammatory responses by activating the p38 and JNK signaling pathways [228,229].The E3 ligase inhibitor of apoptosis protein (IAP) can directly bind to ASK1 and induce its degradation via the E2 enzyme UbcH5.IAP depletion increases TNF receptor 2 (TNFR2)-mediated activation of p38 and JNK, increasing tumor cell proliferation [230].These research findings reveal the role of the UPS in regulating important signaling pathways in various cancers, providing new insights for the future development of cancer therapeutics.

PI3K/AKT/mTOR signaling pathway
AKT, also known as phosphokinase B (PKB), plays a central role in the PI3K/AKT/mTOR signaling pathway [231].Ubiquitin-conjugating enzyme E2S (UBE2S), mitochondrial ubiquitin ligase activator NF-κB (MULAN), ret finger protein 2 (RFP2), breast cancer susceptibility gene 1 (BRCA1), speckle-type POZ protein (SPOP), TNF receptor-associated factor 4 (TRAF4), and F-box and leucine-rich repeat protein 18 (FBXL18), regulate AKT through ubiquitination, affecting its degradation or activation in various cancers (Table 1) [74-76, 78, 79, 232].UBE2S has been shown to be associated with AKT phosphorylation [233].One study found that UBE2S is highly expressed in epithelial ovarian cancer and induces cisplatin resistance by activating the PI3K/AKT/mTOR signaling pathway and inhibiting autophagy.Knocking down of UBE2S can inhibit the proliferation and migration of cisplatin-resistant ovarian cancer cells, providing new insights for the evaluation and treatment of high-risk ovarian cancer patients with cisplatin resistance [234].SPOP, an E3 ligase, inhibits the activity of AKT kinase and its oncogenic function by mediating the ubiquitination and degradation of phosphatidylinositol-dependent protein kinase 1 (PDK1) (upstream protein of AKT).Cancer patients with PDK1 mutations exhibit oncogenic effects by evading SPOP recognition.This could be an attractive therapeutic direction [235].
The mTOR signaling pathway plays a vital role in regulating essential cellular functions such as cell growth, autophagy, metabolism, and DNA damage [236].The lipid phosphatase PTEN can antagonize PI3K [237].In cervical cancer cells, the E3 ligase makorin ring finger protein 1 (MKRN1) ubiquitinates and degrades the PTEN protein.In cervical cancer patients exhibiting high expression levels of MKRN1, the protein level of PTEN is found to be lower, which is associated with a decreased 5-year survival rate [69].Additionally, another study identified that the deubiquitinase OTUD3 interacts with the substrate KPTN to regulate the mTORC1 signaling pathway, significantly inhibiting tumor cell proliferation and growth.By uncovering OTUD3's essential role in cancer, this research provides crucial insights for developing novel cancer treatment strategies targeting OTUD3 or its regulatory pathways [238].Additionally, the E3 ligase FBX8 partially achieves its tumor suppressor function by degrading mTOR in colorectal cancer.Low expression levels of FBX8 are correlated with poor prognosis in colorectal cancer patients [71].In prostate adenocarcinoma (PRAD), the E3 ligase sensitive to apoptosis gene (SAG) targets DEPTOR for degradation, activating the mTORC2/AKT signaling pathway and promoting tumorigenesis.The SAG conditional KO mouse model was employed with PTEN deletion in the prostate to assess the in vivo function of SAG in prostate cancer development, indicating that targeting the SAG E3 ligase could be beneficial in prostate cancer therapy [72].These experiments demonstrate that targeting the E3 ligases that regulate key proteins in the PI3K/AKT/mTOR signaling pathway offers promising therapeutic avenues for various cancers, providing a new direction for developing more effective cancer treatment strategies.

Evading growth suppressors
Inactivation of tumor suppressors eliminates the negative regulation of cell growth and proliferation to promote cancer development [239].In addition to inducing and maintaining positive growth-stimulating factors, cancer cells must evade growth suppressors.Typical tumor suppressors encode retinoblastoma (RB) and p53 proteins, which regulate cell proliferation and apoptosis (Table 1) [6,103].p53 p53 has the highest frequency of mutations in human cancers and is usually expressed at low levels in cancer cells [172].p53 regulates the cell cycle, induces apoptosis in response to DNA damage, and contributes to genomic stability by promoting DNA repair [172].MDM2 functions as a p53 monoubiquitinating E3 ligase, facilitating the ubiquitination and subsequent degradation of p53 [104].Currently, activating p53 by antagonizing MDM2 involves several approaches: (a) reducing MDM2 expression; (b) inhibiting its ubiquitin ligase function; and (c) blocking interactions between MDM2 and p53 [104].The strategy of disrupting MDM2-p53 interactions using small molecules has been extensively pursued.For instance, AMG232 triggered apoptosis and inhibited cell proliferation in glioblastoma and multiple myeloma.The observations also indicated a great specificity for p53 wild-type cells compared to p53 mutant stem cells in glioblastoma [240].AMG232 has also been studied in clinical trials [241].E3 ligases TRIM28 and TRIM31 are also reported to promote p53 degradation.In osteosarcoma cells, TRIM28 cooperates with MDM2 to regulate the ubiquitination and degradation of p53, promoting tumor proliferation [95].A high level of TRIM31 correlates with shorter overall survival (OS) in lung cancer patients [242].Elevated levels of TRIM31 are associated with more aggressive characteristics and unfavorable outcomes in pancreatic cancer patients.Inhibition of TRIM31 increases the sensitivity of gemcitabine in pancreatic cancer cells, indicating suppressing TRIM31 could be an effective approach to improve the efficacy of gemcitabine in overcoming chemotherapy resistance in pancreatic cancer [243].Furthermore, USP7 is identified to directly deubiquitinate p53, inhibiting tumor proliferation.High levels of USP7 and MDM2 are implicated in the onset and development of various cancers, playing a critical role by suppressing p53 activities.Inhibiting these proteins can reactivate p53 pathways, leading to the halting of the cell cycle and programmed cell death.Studies emphasize the pharmacological properties, potential therapeutic uses, and the action mechanisms of small molecule inhibitors targeting USP7 and MDM2 [104].Moreover, USP25 has been shown to be an important upstream regulator of the MDM2-p53 signaling pathway and has the potential to be a novel target gene for developing new therapeutic applications [244].

RB
The dysregulation of the RB pathway is frequently observed in cancer.The impairment of RB function, frequently due to mutations or mechanisms that induce hyperphosphorylation, allows uncontrolled cell cycle progression [245].This process can result in excessive cell proliferation and contribute to tumor development.
In addition to being phosphorylated, RB can be ubiquitinated, sumoylated, acetylated, or methylated [246].TRIM28 binds to the phosphorylated RB protein (p-RB), promoting its ubiquitination and degradation [95].On the other hand, SETDB1, a binding partner of TRIM28, protects p-RB from degradation, which is particularly notable in prostate cancer [247].Inhibiting SETDB1 expression reduces tumor growth but accelerates the degradation of RB protein.Notably, combined use with the CDK4/6 inhibitor palbociclib can block SETDB1 inhibition-induced RB degradation and demonstrate stronger anticancer effects.These research findings reveal the potential value of using a combination strategy of CDK4/6 and SETDB1 inhibition to reduce RB degradation and suppress cancer growth [248].

ADP-ribosylation Factor (ARF)
ARF is a tumor suppressor encoded by the cyclindependent kinase inhibitor 2A (CDKN2A) locus and primarily exerts its tumor suppressive effects through the MDM2-P53 axis [98].Under normal conditions, oncogenic signals induced by MYC, RAS, and E2Fs lead to the upregulation of ARF.ARF subsequently inhibits MDM2, thereby activating the tumor suppressor function of p53 [98,249].ARF function, stability, and cellular localization are tightly regulated by posttranslational modifications such as phosphorylation and ubiquitination [98].Elongin B (ELOB), as a core component of the Cullin2-RBX1-ELOB E3 ligase (CRL2) complex, regulates ubiquitination and degradation of the oncoprotein p14/ARF [250].Research indicates that a peptide strongly adheres to the ELOB/C dimer, disrupting the binding of ELOB/C to its binding molecules.Treatment of cancer cells with this peptide inhibitor led to reduced cell survival, heightened apoptosis, and altered gene activity.Consequently, these findings suggest that targeting the BC-box-binding pocket of ELOB/C is a viable method for disrupting its activity and inhibiting the proliferation of cancer cells [251].Prame is overexpressed in tumor tissues compared to paired adjacent tissues and is associated with poor prognosis in cancer patients.As a substrate recognition receptor protein of Cullin RING E3 ligases (CRLs), Prame regulates the ubiquitination and subsequent degradation of ARF through the Cullin2-RBX1-ELOB E3 ligase complex, making it a potential novel therapeutic target [252].

Resist cell death
Cell death is a normal physiological process in all living organisms and plays essential roles in embryonic development, homeostatic maintenance, aging, and immune coordination [253].Cell death includes apoptosis, necrotizing apoptosis, pyroptosis, autophagy, ferroptosis, cuproptosis [182,253,254].Ubiquitination also plays an essential role in resisting cell death.In the following paragraphs, we will briefly introduce the types of cell death and describe the functions of ubiquitination and deubiquitination in these types of cell death (Fig. 4).

Apoptosis
Apoptosis is the most common noninflammatory form of programmed cell death [182,255].It produces remnants of apoptotic cells, such as cytoplasm, organelles, and the contents of some nuclei, which are randomly sorted into each apoptotic body [105].Two main pathways of apoptosis have been identified: the intrinsic apoptosis pathway and the extrinsic pathway initiated by death receptors [105,182].The intrinsic and extrinsic apoptosis pathways depend on the activation of the caspase family [256].BAX is the main pro-apoptotic executioner protein.The E3 ligase IBR domain containing 2 (IBRDC2) can target BAX for ubiquitination-mediated degradation, thereby preventing mitochondrial outer membrane permeabilization (MOMP) induced by active BAX [105].When inducing apoptosis, IBRDC2 accumulates in BAX-rich mitochondrial structures, allowing the accumulation of BAX to occur simultaneously with its activation [106].BimEL, belonging to the Bcl-2 protein family, crucially promotes apoptosis by inducing mitochondrial outer membrane permeabilization (MOMP) and activating the caspase cascade.The E3 ligase Cbl can degrade the extralong splice variant of Bim (BimEL) and is cell typespecific [105].Icotinib is a specific tyrosine kinase inhibitor (TKI) targeting the epidermal growth factor receptor (EGFR).Treatment with Icotinib significantly reduces the levels of p-EGFR (phosphorylated EGFR), p-ERK (phosphorylated extracellular signal-regulated kinase), and c-Cbl in HCC827 lung cancer cells, leading to inhibited proliferation and induced apoptosis of HCC827 lung cancer cells [257].Aurora A phosphorylates BimEL, enhancing binding to the F-box protein β-transducin, which contains the E3 ligase.This interaction facilitates the ubiquitination and subsequent degradation of BimEL [258].The E3 ligases mentioned above all decrease the levels of pro-apoptotic proteins; therefore, finding drugs that can inhibit the binding affinity of these ligases with their target proteins can maintain a certain level of apoptosis in cancer cells, effectively inhibiting the development of cancer.

Necroptosis
Necroptosis, a type of programmed cell death, involves cell and organelle swelling, membrane rupture, and the release of cellular contents [259].Necrotizing apoptosis is a receptor-interacting protein kinase 1 (RIPK1)-RIPK3mixed lineage kinase domain-like protein (MLKL) pathway triggered by death and Toll-like receptor 3/4 [260].Pellino1 (Peli1) can mediate K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner, promoting the formation of necrosomes and facilitating necroptosis [23].OTULIN can remove the M1 chain from the necroptosis pathway, enhancing TNF-α-induced necrotizing apoptosis [25].Ubiquitin-editing enzyme A20 removes K63-linked ubiquitin chains from RIPK3 [22], inhibiting RIPK3 ubiquitination and reducing RIPK1:RIPK3 interactions.This inhibition effectively restrains TNF-α-induced necrotizing apoptosis, which can be reversed by USP22 [18].Interestingly, several cases of solid tumors with high A20 expression are associated with lower survival rates [261].Knocking down of A20 reduces cell growth and enhances sensitivity to agents that induce apoptosis [262].Moreover, researchers found that A20 plays a vital role in drug resistance, and they established a direct link between elevated A20 levels and increased in vitro resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [263].

Pyroptosis
Pyroptosis is the programmed death of cells caused by the activation of inflammasome sensors.It results in plasma membrane lysis, cell swelling, chromatin fragmentation, and the release of intracellular proinflammatory contents [253,264].Pyroptosis mainly relies on the activation of caspase family proteins by inflammasomes to cause various physiological responses, and NOD-like receptor protein 3 (NLRP3) is a typical inflammasome [265].The E3 ligase TRIM31 can act as a feedback inhibitor for the NLRP3 inflammasome, directly binding to NLRP3, promoting K48-linked polyubiquitination, and facilitating its degradation via the proteasome [96].E3 ligase F-box and leucine rich repeat protein 2 (FBXL2) interact with Trp73 within the NLRP3 protein specifically by targeting the ubiquitination and subsequent degradation of Lys689 [266].The E3 ligase Cbl-b binds to K63 ubiquitin chains on the leucine-rich repeat domain (LRR) of NLRP3 and then targets the K496 site to link with K48 ubiquitin chains and mediate proteasomal degradation [267,268].Notably, by comparing the effects of the NLRP3 inducer Nigericin across various tumor types and normal fibroblast controls, it was discovered that Nigericin may represent a novel therapeutic approach for controlling the growth of tumors that produce low levels of IL-1β and IL-18 [269].According to recent literature, several DUBs were reported to regulate pyroptosis in cancer, including USP18 [270], USP24 [112], and USP48 [113].Mechanistically, USP18 inhibits pyroptosis in cancer cells via enhancing ISGs, while USP48 promotes pyroptosis by stabilizing gasdermin E (GSDEM).An in vivo study indicates that upregulating USP48 can enhance the antitumor activity of PD-1 inhibitor [113], suggesting that USP48 activation pharmacologically could be a promising approach to enhance cancer cell sensitivity to pyroptosis and improve immunotherapy outcomes.

Autophagy
Autophagy is initiated in response to various signals, including nutrient deprivation, the absence of growth factors, hypoxia (low oxygen levels), and exposure to pathogens [271].Autophagy has been shown to play a critical role in tumor maintenance, even with elevated basal autophagy levels in many tumors under nutrient-adequate conditions.Autophagy inhibition or systemic autophagy inhibition in tumor cells disrupts tumor metabolism, resulting in antitumor effects [272].Autophagy can promote tumor cell survival by providing nutrients during periods of stress, such as nutrient deprivation or hypoxia.USP19 plays a significant role in autophagy regulation.It cleaves the ubiquitin chain of NLRP3, inhibiting proteasomal degradation and transforming NLRP3 from a proinflammatory to an anti-inflammatory state [273].Beclin 1 (BECN1) is an important member of the autophagy-related protein family, primarily involved in regulating the formation of autophagic vesicles [109].In various cancers such as breast cancer, ovarian cancer, and colorectal cancer, the expression level of BECN1 is significantly reduced, which is positively correlated with poor prognosis for patients [109].In breast cancer and ovarian cancer, the E3 ligase cullin-3 (CUL3) interacts with BECN1, promoting its K48 ubiquitination and downregulating BECN1, ultimately enhancing tumor cell proliferation and resulting in a poor prognosis [274].Therefore, searching for inhibitors of CUL3 may be an important approach for treating breast cancer and ovarian cancer.A research team has also targeted the functions of Kelch-like (KLHL) family proteins, which are substrate adaptor proteins of Cullin3-RING ligase (CRL3), in order to disrupt the function of CRL3 [97].

Ferroptosis
Ferroptosis is a recently discovered form of cellular death triggered by the excessive accumulation of iron-dependent lipid peroxidation products [275,276].It primarily occurs through exogenous and endogenous pathways involving transporter-dependent and enzyme-regulated mechanisms [276].The regulation of ferroptosis involves a balance between tumor promotion and inhibition.Genetic ablation of solute carrier family seven member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4) induces ferroptosis in cancer cells, leading to significant tumor suppression [277].The E3 ligase TRIM26 mediates the ubiquitination of SLC7A11, targeting it for proteasomal degradation and inducing ferroptosis.This process inhibits hepatic stellate cell activation and reduces liver fibrosis [110].Searching for agonists of TRIM26 might be a strategy for treating early-stage liver cancer.The deubiquitinase BRCA1-associated protein 1 (BAP1) plays an essential role in regulating ferroptosis by removing H2A ubiquitination (H2Aub).BAP1 forms a polycomb repressive deubiquitinase (PR-DUB) complex that deubiquitinates H2Aub on the SLC7A11 promoter, leading to decreased cystine uptake and increased ferroptosis [278].DUB enzyme OTU deubiquitinase ubiquitin aldehydebinding 1 (OTUB1) promotes GPX4 deubiquitination, thereby inhibiting ferroptosis in gastric cancer cells [279].OTUB1 is a distinctive target because of its conventional and unconventional functions.The compound PR-619 is anticipated to decrease OTUB1 activity, as evidenced by its capacity to prevent OTUB1 from binding to an active site probe [114].

Enabling replicative immortality
Telomeres, conserved nucleoprotein structures found at the termini of linear eukaryotic chromosomes, consist of repetitive sequences (TTA GGG )n in humans [280].They interact with six protein species that form a "shelterin complex" [281].As a result of repeated rounds of replication in eukaryotic cells, telomeres are shortened continuously.Therefore, during tumor development, cells must utilize a telomere DNA maintenance mechanism (TMM) to counteract telomere shortening, protect telomeres from the influence of the DNA damage repair system, and avoid telomere-mediated aging and apoptosis [282].Changes in telomere structure are intricately linked to the onset and progression of tumors.Telomere repeats, and the involvement of TRF1 and TRF2 serve as guardians of telomeres, whose expression levels are disrupted across diverse cancer forms [283].In renal cell carcinoma (RCC), the telomere proteins TRF1 and TRF2 are overexpressed, and their inhibition by siRNAs can induce apoptosis, reducing cell proliferation and migration [284].
Recent studies have shown that the ubiquitin mechanism can regulate elements of telomeres.Furthermore, the UPS influences cancer progression by impacting telomeres.Ubiquitin-mediated degradation of telomere associated protein TRF1 levels is facilitated by E3 ligases such as repeatability limit (RLIM), β-TRCP1, and FBX4 (Table 1) [115][116][117].Clinical studies have shown that TRF1 upregulation in glioblastoma multiforme (GBM) contributes to tumor initiation and progression.This was demonstrated by the inhibition of tumor growth and extended survival in GBM mouse models following brain-specific TRF1 genetic deletion.Additionally, chemical inhibitors of TRF1 in human GBM cells blocked tumor sphere formation and slowed growth in patient-derived GSC xenografts [285].These studies suggest the direction of future clinical research on ubiquitination.Conversely, members of the chromatin-modifying complex family, such as general control nonderepressible-5 (GCN5) and USP22, have been reported to facilitate the deubiquitination of TRF1 [286].GCN5 is necessary for the binding of USP22 to Spt-Ada-Gcn5 acetyltransferase (SAGA) complexes, enabling the deubiquitination of TRF1 and preventing its turnover [286].Research has demonstrated that eliminating USP22 from pancreatic tumor cells enhances the immune response by decreasing suppressive myeloid cells and increasing cytotoxic T cells and natural killer cells.Additionally, USP22 influences the cancer cell transcriptome, thereby modifying the immune tumor microenvironment.Targeting USP22 in pancreatic cancer can enhance the effectiveness of immunotherapy and improve treatment outcomes [287].
TPP1, another shelterin protein subunit, also undergoes ubiquitin-mediated proteolysis, which has been evidenced by the stability of TPP1 protein levels after proteasome inhibition.In mice, the stabilization of TPP1 at telomeres requires its ubiquitination by the E3 ligase RNF8 [120].Recent research has indicated that F-box and WD repeat domain-containing 7 (FBW7) can promote cell senescence and tissue fibrosis by facilitating telomere decapitation [118].The deubiquitinase USP7 interacts with human TPP1 and removes ubiquitin chains.Although the degradation of USP7 does not impact the level of TPP1 regulated by the proteasome, USP7 might interact with other deubiquitinases redundantly to stabilize TPP1 [288].By now, P22077 has been extensively studied and has become a prevalent tool compound for inhibiting USP7 in biological research.For example, it has been shown that P22077 can effectively trigger p53-dependent apoptosis in neuroblastoma (NB) cells and markedly reduce tumor growth in xenograft models of three NB cell types [289].Additionally, certain natural compounds have also been identified as USP7 inhibitors.Notably, Spongiacidin C, a pyrrole alkaloid from the marine sponge Stylissa massa, was identified as a USP7 inhibitor with an IC50 of 3.8 μM, though its precise in vivo effects need further investigation [290].

Inducing angiogenesis
Tumor angiogenesis refers to the process of forming new blood vessels within and surrounding tumors.This phenomenon is critical for tumor growth and progression, as it provides the necessary nutrients and oxygen supply to sustain rapidly dividing cancer cells [291].The rapid growth of tumors leads to areas with low oxygen concentrations, known as hypoxia.In response to hypoxia, cells release hypoxia-inducible factors (HIFs) that stimulate the expression of proangiogenic factors, including vascular endothelial growth factor (VEGF), to form new blood vessels [292].The specific prolyl-4-hydroxylase enzyme continuously hydroxylates HIF1α.Once hydroxylated, HIF1α is recognized by the E3 ligase complex, leading to its polyubiquitination and subsequent degradation by the proteasome [291,293].VEGF is highly expressed in most human tumors [294].Under normoxic conditions, the E3 ligase von hippel lindau (VHL) ubiquitinates HIF-1, thereby preventing the dimerization and binding of HIF-1 to the promoter of the VEGF gene and inhibiting its transcription and translation.Under hypoxic conditions, HIF-1 dimerizes and stimulates VEGF production and angiogenesis [295].By regulating the levels of HIF1α, this process directly influences the cellular response to low oxygen conditions.A study has revealed that USP22 promotes the stemness of hepatocellular carcinoma induced by hypoxia-inducible factors through a HIFα/USP22 positive feedback loop after TP53 inactivation [296].This process contributes to promoting angiogenesis, tumor invasion and metastasis, and tumor drug resistance [296].The research team achieved high tumor suppression and increased sensitivity to sorafenib in mice with hepatocellular carcinoma by targeting USP22 with a lipid-polymeric complex.This further indicates that USP22 is a highly promising therapeutic target for hepatocellular carcinoma [296].Additionally, seven in absentia homology 2 (SIAH2) can target HIF for ubiquitination and degradation, thereby modulating the cellular response to hypoxic conditions.HIF-mediated inhibition of nuclear respiratory factor 1 (NRF-1) reduces the transcription of mitochondrial genes and inhibits the activity of the E3 ligase SIAH2 [121].

Activating invasion and metastasis
The invasion and metastasis of tumors include the movement of tumor cells, infiltration into neighboring tissues, circulation, and extravasation to distant organs, which are the leading causes of cancer-mediated damage to the body [297].Ubiquitination and deubiquitination are pivotal in numerous protein modification and regulatory processes and often influence tumor invasion and metastasis.This section aims to explore the role of the ubiquitin mechanism in tumor invasion and metastasis and analyze recent findings related to E3 ligases and DUBs and their potential mechanisms (Fig. 3).

The role of E3 ligases in cancer metastasis
E3 ligases F-Box and WD repeat domain containing 2 (FBXW2) [122], FBW7 [127], Ub-protein ligase E3C (UBE3C) [131], and F-Box protein 22 (FBXO22) [298] play vital roles in cancer metastasis.For instance, FBXW2 functions as a tumor suppressor by facilitating the ubiquitination and degradation of oncogenic proteins such as SKP2 [123] and β-catenin [122], thus impeding cancer migration, invasion, and metastasis.FBXW2 can be ubiquitinated and degraded as a substrate of β-TrCP1 [123].Additionally, overexpression of FBXW2 decreases β-catenin-driven transactivation and suppresses invasion, while depletion enhances β-catenin stability and promotes lung cancer metastasis [122].FBW7 acts as a tumor suppressor by promoting the degradation of cancer-related proteins like Snail [126], Brahma-related gene 1 (Brg1) [127], and YTH N6-methyladenosine RNA Binding Protein F2 (YTHDF2) [128], thereby inhibiting metastasis in various cancers including non-small cell lung cancer, gastric cancer, and ovarian cancer.It also modulates the HIF-1α/CEACAM5 axis in colorectal cancer and potentially predicts immunotherapy response in thymic cancer [129].In clinical studies, it has been shown that low expression of FBW7 in breast cancer cells leads to resistance to the BET inhibitor JQ1, but combining JQ1 with a Mcl-1 inhibitor can overcome this resistance.This finding suggests that enhancing the effectiveness of BET inhibitors in patients with low FBW7 expression is a promising clinical strategy [299].
UBE3C is a tumor promoter that ubiquitinates substrates such as neuroblast differentiation-associated protein (AHNAK), disrupting the p53-AHNAK complex and enhancing stem cell-like properties in non-small cell lung cancer [300,301].It also promotes RCC growth and metastasis by upregulating β-catenin and activating the Wnt/β-catenin pathway [132].In non-small cell lung cancer, FBXO22 promotes Lys63-linked polyubiquitination of liver kinase B1 (LKB1), reducing its activity and impeding the LKB1-AMPK-mTOR pathway, thereby enhancing cell proliferation.Clinically, elevated FBXO22 levels in lung adenocarcinoma patients indicate a poor prognosis [302].FBXO22 promotes angiogenesis and tumor cell migration by increasing the levels of vascular endothelial growth factor A and HIF-1α expression [303].Recent research suggests that FBXO22 may facilitate the ubiquitin-mediated degradation of cyclin G-associated kinase (GAK), thereby inhibiting the proliferation and metastasis of cervical cancer cells [304].Additionally, clinical studies have shown that FBXO22 negativity significantly affects survival in breast cancer patients, especially those with invasive lobular carcinoma (ILC), and leads to poorer outcomes in patients treated with selective estrogen receptor modulators (SERMs) [305].These findings suggest the need for tailored therapeutic strategies based on histopathological types when considering adjuvant endocrine therapy.

The role of DUBs in cancer metastasis
The deubiquitinating enzymes BRCA1-associated Protein 1 (BAP1), USP7, and USP43 are the primary focus of the discussion below.BAP1, characterized by its UCH domain, is a crucial tumor suppressor across various malignancies.In breast cancer, BAP1 promotes tumorigenesis by stabilizing Kruppel-like factor 5 (KLF5) through deubiquitination, facilitating cell cycle progression, while its depletion inhibits tumorigenesis and lung metastasis [306].BAP1 holds significant potential in clinical research.A study on Pembrolizumab efficacy in thymic cancer found that PD-L1 expression, along with alterations in genes or pathways like BAP1, may predict patient response or resistance to immunotherapy [307].USP43 mediates Cav2.2 function by regulating cortical actin stability, extracellular matrix degradation, and migration, with Cav2.2 enhancing USP43 expression through NFAT2 activation, thus promoting breast cancer metastasis [308].USP43 is markedly expressed in epithelial ovarian cancer, fostering cell proliferation, migration, invasion, and cisplatin resistance by stabilizing HDAC2 and activating the Wnt/β-catenin pathway.These discoveries underscore the clinical importance of USP43 in epithelial ovarian cancer, accentuating its potential as a therapeutic target to manage cancer progression, increase sensitivity to cisplatin chemotherapy, and ultimately enhance patient outcomes [309].

Genome instability and mutation
Genome instability is the core of carcinogenesis in multicellular organisms and is characterized by a high frequency of mutations in cell lineage genomes.High-frequency DNA damage and epigenetic or mutation-induced reductions of DNA repair gene expression may contribute to genome instability [310,311].
DDR pathways are complex and intricate.Thousands of endogenous and exogenous DNA damage events occur daily [312,313].The MRE11-RAD50-NBS1 (MRN) complex first recognizes the repair factors recruited at DNA fragmentation sites [314].The E3 ligase RNF126 ubiquitinates meiotic recombination 11 (MRE11) at K339 and K480, activating the DDR and conferring resistance to radiotherapy in triple-negative breast cancer (Table 1) [142].A member of the PI3/PI4-kinase family, ataxia-telangiectasia mutated (ATM) is a protein kinase that is essential for the cellular response to DNA damage, specifically double-strand breaks (DSBs), and is mainly involved in preserving genomic integrity.[315].Research has found that the E3 ubiquitin ligase Peli1 is activated by ATM-mediated phosphorylation, promoting the ubiquitination of NBS1 and enhancing the accumulation of ATM and the MRN complex at DSB sites [316,317].SAM and HD domain containing protein 1 (SAMHD1) combines with the DSB repair initiator CtBP-interacting protein (CtIP) to promote DNA repair [318].It is worth noting that USP7 interacts with SAMHD1 and deubiquitinates the K421 site, thus reducing its degradation by the proteasome to stabilize SAMHD1.Consequently, it repairs DNA damage induced by ROS or genotoxic insults, overcoming carcinogenic stress and influencing chemotherapy sensitivity [145].

Tumor-promoting inflammation
Chronic inflammation is an essential factor in cancer development and is associated with approximately 20% of human cancers [319].Cancer often occurs in inflamed tissues, suggesting that local inflammation plays an essential role in cancer initiation and progression.Moreover, ubiquitination can contribute to tumors initiated by chronic inflammation through the regulation of transcription factors and cytokines, thereby inducing cancer development, maintenance, and metastasis (Fig. 3) [320].
Chronic inflammation and NF-κB activation are closely associated with cancer progression and spread.Linear ubiquitination of key NF-κB regulators by LUBAC plays an essential role.Abnormally regulated linear ubiquitin signaling is associated with cancer initiation and progression [321].For example, elevated LUBAC expression enhances NF-κB activation, accelerating the development of somatic mutations and lymphoma pathogenesis [29].The natural compound thiolutin, which specifically inhibits LUBAC, has been shown to inhibit tumor growth in mouse xenograft models, indicating that LUBAC could be a viable therapeutic target for B-cell lymphoma [29].However, the deubiquitinase OTULIN negatively regulates linear ubiquitin signaling.In hepatocytes, OTULIN deficiency contributes to hepatocellular carcinoma development [152].
In addition, the upstream signaling activator pattern recognition receptor (PRR) is also regulated by E3 ligases [322,323].Toll-like receptors (TLRs) are essential components of the immune system, that can activate NF-κB and induce interferon (IFN) production [323,324].Alternatively, the c-Cbl ubiquitin ligase is involved in TRAF6 ubiquitination and negatively regulates NF-κB activity [325].In the context of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), overexpression of TRIM22 reduces the occurrence and development of endometrial cancer, and its inhibition is mediated by the NOD-NF-κB pathway, which may be one of the mechanisms of NLR [149], indicating TRIM22 may emerge as a valuable prognostic indicator in endometrial cancer patients.TRIM22 can interact with IKKγ, an upstream molecule in the NF-κB pathway, increasing the K63linked polyubiquitination of IKKγ, thereby activating the NF-κB pathway in GBM.This study indicates that inhibiting the E3 ligase activity of TRIM22 or blocking its interaction with the IκBα or IKKγ proteins could have significant implications for the development of potential therapeutic drugs for GBM [326].In addition, NOD1 protects intestinal cells from precancerous lesions by inhibiting the NF-κB signaling pathway through the induction of TRAF3 [150].Cytokines play a crucial role in the tumor microenvironment, promoting communication between malignant cells and surrounding cells.The UPS can influence cancer progression by regulating cytokines [320].For example, SAG plays an important role in chronic inflammation-induced cancers by ubiquitylating key apoptotic factors such as SARM and Noxa, regulating the ratio of pro-and antiapoptotic factors.Therefore, SAG-UPS may serve as an early diagnostic marker for liver cancer and a potential target for therapeutic development [327,328].In conclusion, chronic inflammation, closely linked to aberrant ubiquitination pathways, is a significant driving force in cancer initiation and progression, highlighting potential targets for future cancer treatments and prognostic indicators.

Glucose metabolism
Cancer tissues need to be reprogrammed in terms of both matter and energy to maintain or further enhance the progress of cancer.Otto Warburg was the first to discover the unique metabolism of cancer cells.He observed that cancer cells exhibit a greater tendency toward glycolysis under aerobic conditions, which is called the "Warburg effect" or "aerobic glycolysis" [329].
Hexokinase 2 (HK2) phosphorylates glucose to produce glucose-6-phosphate (G-6-P), regulating glucose metabolism [330].The E3 ligase TRIM36 ubiquitinates HK2 to inhibit the neuroendocrine differentiation (NED) of prostatic cancer (Fig. 5) [153].Additionally, studies have shown that TRIM36 can enhance the efficacy of anti-androgen drugs in treating prostate cancer.Therefore, adding TRIM36 during androgen deprivation therapy (ADT) could be a novel therapeutic approach to better suppress castration-resistant prostatic cancer [331].Recent research has discovered that the E3 ligase membrane-associated RING-CH protein (MARCH8) is a novel glycolysis repressor that inhibits glycolysis in colorectal cancer through the ubiquitination and degradation of HK2 (Fig. 5) [154].However, clinical studies on MARCH8's role in cancer metabolism are lacking, with most research focusing on its involvement in cancer cell apoptosis [332].Future studies could explore therapeutic strategies targeting MARCH8 and its regulatory mechanisms in cancer metabolism, such as combination therapy, to enhance treatment efficacy.In turn, the circular RNA derived from ribosomal protein S19 (circRPS19) upregulates USP7 expression, leading to an increase in HK2 protein levels and the promotion of aerobic glycolysis in gastric cancer cells [165].Therefore, targeting the circRPS19-USP7-HK2 pathway presents a promising therapeutic strategy for treating gastric cancer.PKM2 converts phosphoenolpyruvate (PEP) to pyruvate in the last step of glycolysis.On the one hand, several E3 ligases within the TRIM family, including TRIM9, TRIM29, and TRIM35, have been found to ubiquitinate PKM2 in tumor cells (Fig. 5) [157][158][159].On the other hand, deubiquitinases such as OTUB2, proteasome non-ATPase regulatory subunit 14 (PSMD14) and USP35 enhance the activity and stability of PKM2, thereby promoting glycolysis in tumor cells (Fig. 5) [11,169,170].Additionally, some lncRNAs including the lncRNA LINC01554 and lncRNA UCA1 have been proven to facilitate the ubiquitination of PKM2, thereby suppressing the Warburg effect [333,334].Therefore, developing targeted inhibitors for these enzymes and combining them with traditional chemotherapy could enhance treatment efficacy by addressing multiple aspects of tumor metabolism and growth.In conclusion, ubiquitination plays a crucial role in glucose metabolism by regulating key enzymes.This posttranslational modification influences the activity and stability of enzymes involved in glucose metabolism, impacting the overall cellular energy balance in cancer.

Fatty acid metabolism
Fatty acid metabolism is regulated by three rate-limiting enzymes: ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) [335,336].Peroxisome proliferator-activated receptor γ (PPARγ) is a pivotal regulatory protein that is highly expressed in adipocytes and participates in lipid uptake, synthesis, and storage [337].USP22 upregulates the expression of ACC and ACLY by promoting the K48linked ubiquitination of PPARγ, ultimately fostering lipid accumulation and tumorigenesis in hepatocellular carcinoma cells [172].Furthermore, its overexpression has been linked to a poor prognosis for a number of cancers [338][339][340].This suggests that USP22 could serve as a novel tumor marker for cancer prognosis.The E3 ligase Trib1-COP1 complex targets ACC1 for ubiquitination and degradation, inhibiting leukemia-initiating cells and promoting myeloid differentiation in AML, protecting against leukemia-related mortality and impeding the progression of acute myeloid leukemia (Fig. 5) [162].Hence, identifying a ubiquitination enzyme that counteracts the ubiquitination of Trib1-COP1 is crucial for targeting the fatty acid metabolism pathway for cancer treatment.Another study found that CSN6 can antagonize the activity of the E3 ligase FBXW7β, preventing FBXW7β-mediated ubiquitination and degradation of FASN, thereby positively regulating lipogenesis in colorectal cancer (Fig. 5) [163].Importantly, the study demonstrated that combining cetuximab with orlistat can inhibit the growth of CSN6-high patient-derived xenograft (PDX) tumors.These findings hold key prognostic and therapeutic significance for colorectal cancer patients [163].On the other hand, USP38 can deubiquitinate and stabilize FASN in gastric cancer, increasing triglyceride production and promoting growth and migration in gastric cancer cells [204].In addition, orilistat, an inhibitor of USP38, can reverse the phenotype of USP38 overexpressed gastric cancer cells [204].Therefore, targeting FASN with USP38 inhibitors can be used as a potential treatment for gastric cancer patients with high expression of USP38.Moreover, previous reports have indicated that FASN serves as a substrate for USP14 in hepatocytes, but other reports suggest that FASN levels in cancer cells are not considerably impacted by the USP14 inhibitor IU1, suggesting that FASN may not be a Fig. 5 The ubiquitin-proteasome system regulates tumor metabolism in several pathways.a Glycolysis: Hexokinase 2 (HK2) can be ubiquitinated by E3 ligases membrane-associated RING-CH protein 8 (MARCH8) and tripartite motif protein 36 (TRIM36), a process that can be reversed by the deubiquitinase ubiquitin-specific protease 7 (USP7).Pyruvate kinase M2 (PKM2) can be ubiquitinated by E3 ligases TRIM9, TRIM29, STIP1 homology and U-box-containing protein 1 (STUB1), and TRIM35.Conversely, PKM2 can be deubiquitinated by proteasome non-ATPase regulatory subunit 14 (PSMD14), OTU domain-containing ubiquitin aldehyde-binding protein 2 (OTUB2), and USP35.b Fatty acid (FA) metabolism: Acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), both involved in fatty acid metabolism, can be ubiquitinated by E3 ligases tribbles pseudokinase 1 (Trib1)-constitutive photomorphogenic 1 (COP1), and F-box and WD repeat domain containing 7β (FBXW7β), respectively.In contrast, FASN can be deubiquitinated by USP38.c Glutaminolysis: Glutaminase C (GAC), which catalyzes the initial step of glutamine decomposition into glutamic acid and ammonia, can be ubiquitinated by the E3 ligase TRIM21.This leads to K63-linked ubiquitination that inhibits GAC activity.Glutamate dehydrogenase (GDH), another key enzyme in glutamine catabolism, can be ubiquitinated by E3 ligases ring finger protein 213 (RNF213) and STUB1 direct substrate of USP14 in cancer cells [341,342].Given the above, further investigations are needed to clarify the functions of the ubiquitination and deubiquitination of pivotal enzymes in fatty acid metabolism.

Amino acid metabolism
Amino acid metabolism in cancer is influenced by ubiquitination and deubiquitination.Glutamate dehydrogenase (GDH) catalyzes the deamination of glutamic acid and has two subtypes, GDH1 and GDH2, of which GDH1 is mainly degraded by the ubiquitin-proteasome pathway [343].The E3 ligase RNF213 mediates GDH1 degradation in kidney renal clear cell carcinoma (KIRC) (Fig. 5) [161].Researchers found that the loss of GDH1 promotes tumor formation after amino acid deprivation by reducing α-ketoglutarate (αKG) levels and αKG-dependent lysine demethylase (KDM) activity [161].Additionally, another study in hepatocellular carcinoma identified two GDH1 inhibitors: Quercetin and Permethylated Anigopreissin A [344].We can hypothesize that applying these drugs to KIRC could maintain αKG levels and KDM activity, potentially preventing the progression of KIRC.The only enzyme in mammals capable of eliminating ammonia and glutamic acid and synthesizing glutamine de novo is glutamine synthetase (GS) [345].In multiple myeloma (MM), USP15 controls the ubiquitination of GS, which is mediated by the E3 ligase complex Cul4-DDB1-CRBN-RBX1 (CRL4 CRBN ) [171].Interestingly, immunomodulatory drug (IMiD)-resistant cells have high expression of USP15, and lenalidomide, an immunomodulatory medication, can sensitize these cells when USP15 is depleted [171].Thus, focusing on USP15 offers a significant therapeutic potential to improve the efficacy of CRBN-based PROTAC treatments for the treatment of cancer.A preclinical study mentioned a small molecule inhibitor of USP15 (USP15-Inh) provided by Forma Therapeutics [346].Glutaminase (GAC) catalyzes the initial step of glutamine decomposition, converting it into glutamic acid and ammonia [347].The E3 ligase TRIM21 promotes K63-linked ubiquitination of GAC, inhibiting its activity in non-small cell lung cancer (Fig. 5) [164].Furthermore, acetylation of Lys311 on GAC further enhances this inhibitory process, thereby suppressing non-small cell lung cancer progression and offering new insights for targeting TRIM21 in lung cancer therapy [164].

Evading immune destruction The crosstalk between the ubiquitin protein system and the TME
Tumors are closely related to the surrounding microenvironment and constantly interact with each other [348].Ubiquitination is a common posttranslational modification that plays a vital role in regulating cellular signal transduction pathways in the TME [320].This modification effectively stimulates antitumor immunity and modulates the balance between tumor suppressors and oncoproteins by modulating the immune response [349].The function of the UPS is to influence the TME by directly or indirectly regulating the degradation of immune checkpoint molecules and the release of oncogenic cytokines [8].In conclusion, TME is an important component affecting tumor growth and development.The UPS affects tumor progression by regulating the interaction between tumors and the TME (Fig. 3).

The role of ubiquitination in crosstalk between CAFs and tumor cells
Cancer-associated fibroblasts (CAFs) are the most common cells in the TME.CAFs regulate the activities of tumor cells and other stromal cells through direct contact and by secreting regulatory factors, especially TGFβ, IL-6, and CC-chemokine ligand 2 (CCL2).Therefore, CAFs play an essential role in tumor progression [350].
TGF-β plays a vital role in the epithelial mesenchymal transition (EMT), promoting the transition of epithelial cells to motile mesenchymal cells and thereby promoting the migration and invasion of tumor cells (Fig. 6) [351].The USP family is involved in regulating the TGFβ-induced EMT.According to recent literature, USP3, USP4, USP11, USP15, and USP26 positively regulated TGF-β signaling in various cancer types [194,[197][198][199].For example, USP11 regulates TGF-β-induced plasticity and promotes breast cancer metastasis by stabilizing TGF-βRII [196].High expression of USP11 was found in gastric cancer patients' tumor samples, and its upregulation promoted gastric cancer tumor growth and metastasis.Interestingly, suppression of USP11 enhanced the sensitivity of GC cells to chemotherapy [352].Additionally, in human basal-like basal cells, overexpression of the ubiquitin-editing enzyme A20 amplifies the TGF-β1induced epithelial-mesenchymal transition by enhancing the polyubiquitination of Snail1.Knockdown of A20 reduces cancer metastasis in mouse xenograft tumors and an orthotopic breast cancer model, suggesting that the polymononubiquitination of A20 and Snail1 plays a key role in the metastasis process [184].In addition, the DUB USP27X is regulated by TGF-β during the EMT and maintains the stability of Snail1 in breast cancer and prostate cancer.Inhibition of USP27X leads to the destabilization of Snail1, inhibits the EMT process, and enhances the sensitivity of tumor cells to chemotherapy [200].

The role of the ubiquitin protein system in immune checkpoints
Increasing evidence has shown that the UPS plays an important role in immune checkpoints [353].The primary focus is on the role of the UPS in the PD-1/ PD-L1 pathway [8].These findings suggest that the UPS is a novel approach for enhancing antitumor immunity.E3 ligases and deubiquitinases are pivotal in modulating the stability of PD-1 and PD-L1 [176].SPOP proteins regulate various cancer-related substrates and play a crucial role in mediating PD-L1 degradation [354].Research has found that CDK4/6 increases PD-L1 protein levels by inhibiting the phosphorylation of SPOP mediated by cyclin D-CDK4.The combination of CDK4/6 inhibitors with anti-PD-1 immunotherapy has been shown to reduce tumors and significantly improve overall survival rates in mouse tumor models [173].Additionally, as a subunit of the SCF E3 ligase complex, the F-box protein plays various roles in human tumors, including mediating the ubiquitination of PD-1 [176,177].One experiment showed that FBXO22 could activate the ubiquitination of PD-L1 to increase the sensitivity of non-small cell lung cancer cells to DNA damage, with CDK5 acting as an upstream regulator of FBXO22.Research suggests that combining CDK5 inhibitors with immune checkpoint inhibitors enhances the efficacy of immune checkpoint blockade therapy [178].In addition, USP22, and USP9X stabilize PD-L1 through deubiquitination, promoting cancer development and migration [187,193,355].Studies also show a positive correlation between PD-L1 and USP12 levels, with USP12 knockout desensitizing mouse lung tumor cells to anti-PD-1 therapy [192].Additionally, USP14 knockout or inhibition enhances cell response to PD-1 inhibitors [191].UPS also plays a vital role in cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) [185].TRAF6 is involved in CD8 + T-cell-mediated antitumor immunity by promoting the ubiquitination and degradation of CTLA-4 [185].The role of the UPS in immune checkpoints, particularly the PD-1/PD-L1 pathway, suggests a potential for novel therapeutic strategies aimed at enhancing antitumor immunity.irreversible growth cessation.This process acts as a barrier to the sustained proliferation needed for tumor formation.Phenotypic plasticity can lead to changes such as dedifferentiation, blocked terminal differentiation, and transdifferentiation [356].Subsequently, the impact of ubiquitination on surface plasticity was explored through an examination of its effects on dedifferentiation and transdifferentiation, along with a discussion of the processes of ubiquitination and deubiquitination.

During organ development, cells organize into tissues and undergo terminal differentiation, often leading to
The first step is dedifferentiation from the mature state to the progenitor cell state.Dedifferentiated liposarcoma (DDLPS) is an invasive adipose cell carcinoma.Characterized by a low tumor mutation burden and frequent chromosomal structural abnormalities, DDLPS often exhibits amplification of the MDM2 gene.MDM2, an E3 ligase, is responsible for targeting the degradation of p53.The overexpression of MDM2 in human cancers has been associated with a poor prognosis [202].Targeting MDM2 is a promising therapeutic strategy, as demonstrated by the growing number of MDM2 inhibitors, such as RG7112, AMG-232, and APG-115, undergoing clinical trials [357].Second, transdifferentiation refers to the transformation of tissue cells into cells of different lineages.The ubiquitin proteasome system is involved in the transdifferentiation process of specific cancers.The transformation of adventitial fibroblasts (AFs) into myofibroblasts is pivotal in the vascular restructuring observed in conditions such as atherosclerosis, restenosis, and aortic aneurysm.Notably, NADPH oxidase 4 (Nox4) undergoes ubiquitination through direct engagement with the ubiquitin-specific protease domain of CYLD.Elevated levels of CYLD and Nox4 in the adventitia due to hyperhomocysteinemia significantly enhance AF transformation, exacerbating CaPO 4 -induced abdominal aortic aneurysm progression in mice [203].Furthermore, neuroendocrine prostate cancer represents a deadly subtype of prostatic cancer that is distinguished by the attenuation of AR signaling during neuroendocrine transdifferentiation.This alteration ultimately contributes to the development of drug resistance to AR-targeted therapies.Researchers have shown that the differentially expressed gene Rac GTPase-activating protein 1 (RACGAP1) is involved in the NE transdifferentiation of prostatic cancer.The underlying mechanism is that RACGAP1 promotes the neuroendocrine transdifferentiation of prostatic cancer by stabilizing the expression of EZH2 in the ubiquitin proteasome pathway [358].

Nonmutational epigenetic reprogramming
In recent years, as research on chromatin and histones has deepened, epigenetics has gradually emerged.Epigenetics encompasses diverse modifications in gene expression that occur without modifying the gene's DNA sequence, resulting in hereditary changes in the gene's function [359,360].Epigenetic phenomena include DNA methylation, histone modification, chromatin remodeling, and noncoding RNAs [361,362].Studies on epigenetic phenomena related to ubiquitination and deubiquitination include DNA methylation, histone modification, and chromatin remodeling (Fig. 3).

DNA methylation
DNA hypermethylation of the promoter at the cytosinephosphate-guanine (CpG) sequence is a clearly defined epigenetic marker in all human tumor types and leads to the silencing of tumor suppressor genes (TSGs) and other genes associated with cancer, thus giving precancerous cells a selective advantage [363,364].Recent research on ubiquitination and deubiquitination in DNA methylation focuses on DNA methyltransferases (DNMTs).The E3 ligase RNF180 ubiquitinates DNMT1, significantly reducing PCDH10 methylation levels and increasing its expression in gastric cancer.Furthermore, the positive co-expression of RNF180 and PCDH10 is associated with a favorable clinical prognosis in gastric cancer patients, suggesting that PCDH10 and RNF180 could be potential biomarkers for gastric cancer diagnosis [206].A similar study revealed the RNF180/DNMT3A/ADAMTS9 axis in gastric cancer.ADAMTS9 significantly inhibits cell viability and motility both in vitro and in vivo.RNF180 ubiquitinates DNMT3A, markedly reducing ADAMTS9 methylation levels and increasing its expression in gastric cancer [207].Therefore, finding an inhibitor that targets RNF180 could potentially inhibit the progression of gastric cancer from multiple angles.Fructose-1,6-bisphosphatase 1 (FBP1) is an enzyme that catalyzes a key step in gluconeogenesis, converting fructose-1,6-bisphosphate to fructose-6-phosphate [365].Nuclear FBP1 has been found to interact with DNMT1 and to recruit PARP1 to chromatin, enhancing the sensitivity of pancreatic cancer to the poly ADP-ribose polymerase (PARP) inhibitor Olaparib [211].Significantly, USP7 can reverse this by deubiquitinating FBP1, thereby inhibiting this interaction.Consequently, USP7 inhibitors enhanced the anti-tumor effects of PARP inhibitors in an FBP1-dependent manner.Therefore, combining USP7 inhibitors with PARP inhibitors might yield a more potent anti-tumor response than using PARP inhibitors alone, potentially offering a more effective treatment approach for PC [211].

Histone ubiquitination
Histone octamer is composed of H2A, H2B, H3, and H4, which are connected with DNA to form nucleosomes [366].Histone ubiquitination contributes to proper DSB repair and plays a significant role in the interaction with transcription and replication [367].The E3 ligase RNF40 can interact with the E3 ligase complex DNA binding protein 1 (DDB1)-Cullin 4a (CUL4A), inhibiting the ubiquitination of H2A by DDB1-CUL4A [208].Notably, RNA demethylase ALKB Homolog 5 (ALKBH5)-mediated m6A deficiency in osteosarcoma leads to increased expression of USP22 and RNF40, suppressing H2A ubiquitination and promoting gene expression related to replication and DNA repair, driving osteosarcoma progression [208].Therapeutic strategies could focus on creating modulators to enhance or mimic ALKBH5 activity, targeting the overexpression of USP22 and RNF40, offering a new potential treatment for cancers with m6A abnormalities.Cyclin B1, encoded by the CCNB1 gene, is regulated by H2B ubiquitination at its promoter by the E2 enzyme radiation sensitive 6 (RAD6) in esophageal squamous cell carcinoma (ESCC), affecting ESCC cell proliferation [205,368].Additionally, a novel RAD6 selective small molecule inhibitor targeting the catalytic site of RAD6 (SMI#9) enhanced the sensitivity of cancer cells resistant to cisplatin or oxaliplatin in triple-negative breast cancer and colorectal cancer [369].Using SMI#9 could provide a strategy to overcome drug in chemotherapy, offering a promising avenue for enhancing the effectiveness of existing cancer treatments in various resistant tumors.Therefore, specific human proteins can affect histone ubiquitination by regulating E3 ligases or E2 ubiquitin binding enzymes, impacting the cell cycle and cancer development.Refer to section II.A for additional details on histone ubiquitination.

Chromatin remodeling
Nucleosomes, linker histones, and nonhistones undergo further assembly into highly organized chromatin structures, restricting access to DNA [370].The main subfamilies of chromatin remodeling complexes are switch/ sucrose nonfermentable (SWI/SNF), imitation SWI (ISWI), chromodomain-helicase DNA-binding protein (CHD), and inositol-requiring mutant 80 (INO80) [371,372].The SWI/SNF complex was the first remodeling complex discovered [373].In prostate cancer, OTUD6A deubiquitinates the SWI/SNF ATPase subunit Brg1, promoting cancer progression [212].AT-rich interactive domain protein 1A (ARID1A), a SWI/SNF complex component, acts as a tumor suppressor [374].In gastric cancer and hepatocellular carcinoma, the E3 ubiquitin ligase complex SCF promotes degradation of ARID1A, which is triggered by ATM activation due to DNA damage in gastric cancer and by mTORC1 activation in hepatocellular carcinoma [186,209].Some progress has been made in the clinical application of targeted anticancer therapies focusing on SCF complexes, particularly through inhibitors of cullin neddylation and Skp2, such as MLN4924 and Erioflorin [375,376].During normal DNA synthesis, BAP1 stabilizes INO80 through deubiquitination.Additionally, BAP1 recruits INO80 to replication forks by interacting with H2Aub, thereby facilitating fork progression.This process underscores the molecular basis of BAP1's tumor suppressor function [377].Additionally, recent research indicates that the E3 ligase CHIP also can stabilize INO80 through nondegradable ubiquitination.Therefore, CHIP and BAP1 collaborate to regulate the ubiquitination of INO80, thereby facilitating DNA replication [210].These findings are noteworthy for the investigation of chromatin remodeling in cancer.

Polymorphic microbiomes
Increasing evidence suggests that the polymorphic variability of the microbiome between individuals impacts cancer phenotypes, and distinct characteristic microbiota have been recognized in different tumors [378,379].Micromonas infection can enhance the proliferation and inflammatory response of colorectal cancer cells, and Parvimonas micra (P.micra) was found to affect protein expression in colorectal cancer intestinal epithelial cells.The upregulation of proteasome β4 (PSMB4) protein indicates the critical role of the ubiquitin proteasome pathway in colorectal cancer [213].In addition, propionate, a microbial metabolite, was shown to target euchromatic histone lysine methyltransferase 2 (EHMT2) by coordinating proteasomal degradation through the upregulation of HECT domain E3 ubiquitin protein ligase 2 (HECTD2).Propionate treatment initially increases the expression of HECTD2, which then facilitates the proteasomal degradation of EHMT2 through posttranslational modification.EHMT2, through H3K9 dimethylation, forms heterochromatin structures and negatively regulates tumor necrosis factor alphainduced protein 1 (TNFAIP1) [214].Consequently, the degradation of EHMT2 reduces H3K9 dimethylation in the TNFAIP1 promoter region, leading to the upregulation of TNFAIP1 and apoptosis of colorectal cancer cells [214].Furthermore, treatment of colorectal cancer cells with Clostridium butyricum (C.butyricum) decreased MYC-mediated resistance to 5-FU and enhanced the effectiveness of anti-PD-1 immunotherapy [380].In summary, the microbiota and its metabolites can regulate tumor development and therapeutic efficacy by affecting the UPS.These experiments suggest potential therapeutic strategies and highlight the importance of microbiota mechanisms of action in cancer research.

Senescent cells
Senescence is an irreversible state of cell cycle arrest that occurs when cells respond to various stress factors, including DNA damage or activation of oncogenes [381].While senescence can exert a tumor suppressive effect by preventing the proliferation of damaged or mutated cells, it also has potential impacts on the development and progression of cancer [7].Senescence in cancer may rely on oncogene-induced senescence (OIS) and tumor suppressor gene loss-induced senescence (TIS).The P16 INK4A -RB and p53-p21-RB pathways are crucial mechanisms for initiating and maintaining growth arrest [381,382].Melanoma is a malignant tumor that originates from melanocytes in the skin and is rich in senescent cells.In melanoma, overexpression of mutant promotes excessive proliferation of melanocytes, inducing the expression of P16 INK4A , which subsequently inhibits the activity of CDK4 and CDK6, leading to hypophosphorylation of RB, cell cycle arrest, and thus inhibiting cancer development [383].In a study, primary mouse and human cells lacking BRCA2 exhibited senescence characteristics, which reversed upon the loss of ARF [98].This may be due to the ability of ARF to inhibit the activity of E3 ligase MDM2, thus enabling normal p53 function.Targeting the interaction between MDM2 or ARF-MDM2 using small molecule inhibitors may help restore p53 function in the impaired p53 signaling pathway, promoting senescence or apoptosis in cancer cells [384].ALRN-6924 reactivates p53 function by inhibiting two proteins, MDM2 and MDMX, subsequently inhibiting tumor cell growth [384].Compounds like ALRN-6924, which induce inflammatory responses and reduce immune evasion in the tumor microenvironment, could be effectively combined with immunotherapies, especially in melanomas rich in senescent cells [384].Exploring the interplay between ubiquitination, deubiquitination, and cellular senescence opens promising avenues for developing targeted therapies that can selectively modulate cell fate in cancer.

Proteinase inhibitors
The proteasome is a highly anticipated target in cancer therapy.Proteasome inhibitors (PIs), like bortezomib, carfilzomib, oprozomib, and ixazomib, have been successfully developed for clinical treatment and have shown good efficacy (Fig. 7) [184,385,386].
Bortezomib is used to treat patients with MM by inhibiting the chymotrypsin-like activity of the 26S proteasome [387,388].Although its use alone may cause a wide range of unintended side effects, its toxicity can be reduced when used in combination with pomalidomide and dexamethasone [389,390].In addition, the FDA has approved bortezomib for treating mantle cell lymphoma [391].Currently, bortezomib is in clinical trials for treating other types of cancer, such as autoimmune hemolysis and COAD [392,393].Carfilzomib is a tetrapeptide epoxide that selectively binds to the 26S proteasome and inhibits protease activity.It was approved by the FDA in 2012 for the treatment of multiple myeloma [394,395].Carfilzomib is undergoing clinical trials for various cancers, including renal cell carcinoma, lymphoma, acute myeloid leukemia, lymphocytic leukemia, and small-cell lung cancer.
Oprozomib is a derivative of carfilzomib designed to have better oral bioavailability.Oprozomib has shown similar antitumor activity and efficacy to carfilzomib in the treatment of multiple myeloma.Therefore, it can be used for the treatment of multiple myeloma resistant to bortezomib, dexamethasone, or lenalidomide [396].Oprozomib induces apoptosis by upregulating the proapoptotic proteins Bcl-2 interacting killer (BIK) and MCL-1.It can be used to treat solid tumors [397].However, oprozomib has high gastrointestinal toxicity and unstable pharmacokinetics.
Ixazomib was used to inhibit the activity of the 20S proteasome [398,399] and its antitumor effect is superior to bortezomib [399].Ixazomib combined with lenalidomide/dexamethasone significantly improves the survival of patients with myeloma [400].Ixazomib has completed phase I clinical trials for glioblastoma multiforme (GBM) and phase II clinical trials for malignant myeloid and lymphatic blood cancers.The patients who exhibited resistance to bortezomib demonstrated a favorable response to isazomil.In addition to these inhibitors, other proteasome inhibitors, such as marizomib and delanzomib, are also undergoing clinical trials.For example, some studies have tested the effects of malizomil on glioblastoma through its ability to cross the blood brain barrier [395].

E1 and E2 inhibitors
The E1 enzyme functions to activate ubiquitin and transfer it to the E2 enzyme [1].Adenosine 3' , 5' monophosphate (AMP) is tightly bound to the E1 enzyme in the catalytic cascade of ubiquitin activation, and some AMPrelated drugs can act as E1 inhibitors.The major sodium adenosulfonate E1 inhibitors include perazone, TAK-243, ML-792, TAK-981, acetyl-DL-carnitine, ABPA3, and ABP1 (Fig. 7) [401].Among the E1 inhibitors in clinical trials, the only one with published clinical trial data is pevonedistat.Combination therapy with risperidone, such as risperidone and azacitidine, has shown more promising efficacy in the treatment of AML patients, and carboplatin and paclitaxel have shown better clinical benefits in the treatment of advanced solid tumors.In patients with AML with TP53 mutations, the composite CR/PR rate was 80% with pevonedistat and azacitidine combination therapy [402,403].
E2 enzymes primarily facilitate the binding of ubiquitin to substrates.Current efforts are focused on identifying inhibitors that disrupt the interaction between E1 enzymes and E2 enzymes or between E2 enzymes and E3 ligases.For instance, Leucetta A has been shown to inhibit the interaction between ubiquitin-conjugating enzyme 13 (UBC13) and ubiquitin-like protein one activating enzyme (UEV1A), thereby preventing complex formation [404].Alternatively, manadosterols A and B, isolated from the sponge Lissodendoryx fibrosa, target the same molecular interaction as Ubc13-Uev1A [405].In addition, the E2 enzyme Cdc34 inhibitor CC0651 blocks the ubiquitination and degradation of p27, thereby inhibiting tumor cell proliferation [406].

E3 inhibitors
The E3 ligase interacts with the ubiquitin-activating enzyme E1 and the ubiquitin-conjugating enzyme E2 to complete the ubiquitination process [407].Drugs targeting E3 ligases play an essential role in cancer therapy by linking ubiquitin to specific protein amino acids.Clinical research information on these inhibitors has been obtained from https:// clini caltr ials.gov/ and https:// pubch em.ncbi.nlm.nih.gov/, as listed in Table 2.

PROTACs
Small molecule drugs are designed to specifically target disease-related proteins using lock-and-key mechanisms.This approach relies on the presence of suitable pocket regions in the target protein structure as small molecule binding sites.PROTAC technology provides an essential platform for inducing the degradation of target proteins.The PROTAC molecule consists of two components, a ligand capable of specifically binding to the target protein and a ligand that recruits an E3 ligase to promote ubiquitination of the captured protein, leading to target protein degradation [441,442].ARV-110 and ARV-471 have progressed to phase II clinical trials.Much of the subsequent discussion has focused on these two pharmaceutical compounds.
ARV-471 is an oral heterobifunctional molecule that uses PROTAC technology to target estrogen receptor (ER) α and has potential antitumor activity [443].A phase I study involving patients with ER + and HER2-BC found that ARV-471 significantly reduced ER expression by up to 90% in tumor tissue.The phase I data indicated that ARV-471 performs well at any dose, substantially degrades ER, and is well tolerated.ARV-471 degrades both wild-type and mutant ER proteins.ARV-471 is undergoing a phase II clinical trial evaluating its efficacy in patients with ER + /HER2 + locally advanced and metastatic breast cancer [444,445].Oral ARV-471 monotherapy showed promising antitumor activity in estrogen-dependent MCF7 xenografts and a significant reduction in ER protein levels.Enhanced anticancer effects were noted when combined with the CDK4/6 inhibitor, palbociclib [446].ARV-471 also showed a good inhibitory effect on a hormone-independent PDX model of estrogen receptor 1 (ESR1) mutants [447].
These results demonstrate the feasibility of the PROTAC approach in patients.

Molecular glue
Although PROTAC technology offers significant potential for drug development, the designed molecules are typically large.An alternative effective strategy involves the use of molecular glue degraders, small molecules capable of facilitating novel interactions between target proteins and E3, ultimately leading to ubiquitinationmediated degradation of the target protein [448].Unlike PROTACs, molecular glues have better chemical properties and smaller molecular volumes (Table 3).
Progress has been made in the development of drugs containing the E1 and E2 enzymes.However, because E3 ligases can bind to target proteins more precisely and specifically, drugs that act on E3 ligases are expected to be developed [458].CC-90009 can recruit SPT1 to the CRL4 CRBN E3 complex and promote the ubiquitination of GSP for proteasomal degradation [13].Serdemetan (JNJ-26854165) is an antagonist of the human double minute 2 (HDM2) E3 ligase, that blocks p53 degradation by inhibiting the ubiquitination of HDM2.In addition, serdemetan can inhibit cholesterol transport.Clinical research on human cell lymphoma and multiple leiomyomas is underway [454,459].Notably, several small molecule drugs targeting MDM2 have been identified, as shown in Table 3.In addition, phase I clinical trials are currently underway for KPG-818, a potential therapeutic agent targeting Cullin-RING ligase 4 (CRL4) to treat hematological malignancies (NCT04283097) [460].
HECT-type E3 ligase inhibitors Research indicates that heclin, a small molecule inhibitor, can alter the conformation of the HECT domain, significantly suppressing the activity of HECT-type E3 ligases and demonstrating antitumor properties [465].In addition, a high-throughput screen identified that clomipramine, an inhibitor of the HECT ubiquitin E3 ligase ITCH, acts as an autophagy modulator to inhibit the growth of breast cancer, prostate adenocarcinoma, and bladder urothelial carcinoma cells [466].Alternatively, a molecular model of the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) inhibitor complex, which combines saturation transfer differential nuclear magnetic resonance (STD NMR), DEEP-STD NMR methods, and docking calculations, has recently been proposed to provide a method for the development of novel inhibitors [467].
IAP inhibitors IAP inhibitors were created by mimicking Smac/Diablo, a natural antagonist of IAPs, to induce the proteasome-dependent degradation of cIAP1, cIAP2, and X-linked IAPs [474].Various small molecule inhibitors targeting the IAP are clinically available, as shown in Table 2

DUB inhibitors
Ubiquitination is a dynamic process managed by DUBs, which facilitate the removal and alteration of UB or polyubiquitin chains from ubiquitinated proteins [476].Many DUBs are involved in the cell cycle process, regulation of genomic instability, and various events in tumorigenesis [477].As a result, numerous inhibitors of DUBs have been developed, including both broad-spectrum and targeted varieties, all of which are recognized as promising candidates for cancer therapy [478,479].G5 and F6 are broad-spectrum DUB inhibitors discovered through cell-based drug screening [480].These chalcone DUB inhibitors are known for their ability to induce apoptosis in BCL-2-independent cells [480,481].Through active chemical proteomics, compound PR619 was suggested as a broad-spectrum DUB inhibitor [482].NSC632839, another potent inhibitor with broad-spectrum activity against deubiquitinases, selectively targets USP2 and USP7, inducing apoptosis in cancer cells [480].As a specific USP1 inhibitor, pimozide can block the maintenance and radiation resistance of glioma stem cells [483].WP1130 inhibits several DUBs, including USP9X, USP5, USP14, ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5), and UCH37.It decreases the MCL-1 level and increases the p53 level, showing antitumor effects [484].Betulinic acid, derived from various plants, has been recognized as a broad-spectrum DUB inhibitor, that triggers an aberrant transmembrane potential and apoptosis in cancer cells [485,486].Nevertheless, these inhibitors might amplify their impacts and nonspecific toxicity through diverse mechanisms, underscoring the clinical preference for specific DUB inhibitors [487].
USP7 is widely recognized as a target for drug development due to its key role in regulating p53 stability.USP7 antagonists, such as p5091 and p50429, have been developed to promote the ubiquitination and degradation of MDM2, leading to bortezomib resistance in MM cells [482,488,489].Moreover, FT671 and FT827 target dynamic pockets near the USP7 catalytic center via selfinhibiting apolipoproteins [490], thereby disrupting the stability of the USP7 substrate, increasing the protein levels of p53 and its related genes, and ultimately inhibiting tumor growth [490,491].A series of small molecules, including HBX 19818, HBX28,258, P22077, and P50429, were shown by biochemical tests and protein mass spectrometry to have specific inhibitory effects on USP7 [489,492].USP14, which is related to WNT/β-catenin signal transduction [493,494], is overexpressed in various cancer types and is positively associated with a poor prognosis [495,496].The inhibitor IU1 can effectively inhibit the activity of the USP14 enzyme by blocking its binding to the proteasome and enhancing its function [497].
Various new screening methods have been used to identify inhibitors and related compounds that target DUBs.For instance, high-throughput screening techniques were employed to identify selective inhibitors targeting UCH-L1, resulting in the identification of LDN-57444.This compound has been shown to trigger apoptosis in lung cell lines [498].Furthermore, a cell-based screening methodology has been employed to identify compounds that can induce apoptosis across various tissue types, with b-AP15 serving as a prominent example [499].B-AP15 triggers the accumulation of high molecular weight ubiquitin (Ub) complexes within cells and acts as an inhibitor of 19S regulatory particles.It selectively targets the ubiquitination activity of USP14 and other ubiquitin enzymes without impacting proteasome function [500].In addition, b-AP15 inhibits the degradation of proteasome substrate proteins, leading to the accumulation of ubiquitin, which induces significant protein stress and mitochondrial damage [501,502].In various solid tumors and multiple myeloma, it may lead to tumor apoptosis through a c-MYC-NoXa-mediated pathway [500,501,503].

Conclusion
In the past few decades, significant progress has been achieved in the study of the UPS.In this review, we comprehensively reviewed the research progress on the UPS regarding tumor characteristics and treatment strategies.Regarding tumor characteristics, the specific mechanisms by which ubiquitination influences cancer progression remain unclear.Therefore, future investigations must delve into the specific mechanisms involved and endeavor to elucidate the efficacy of the UPS.In terms of treatment strategies, although we have collected some clinical drug information, we also noticed that some knowledge gaps persist, especially regarding E1-and E2-targeted drugs.These findings suggest that future drug research should focus more on these aspects.
Based on the role of the UPS in cancer, potential therapeutic targets have been identified, and the corresponding inhibitors have been further studied.Proteasome inhibitors, such as bortezomib, carfilzomib, oprozomib, and ixazomib, have been approved by the FDA.They have achieved good clinical results, but their widespread application is limited by the side effects caused by the abnormal accumulation of some upstream proteins.Therefore, targeted inhibitors of E1 enzymes, E2 enzymes, E3 ligases, deubiquitinases, and other targets, including MDM2 inhibitors, IAPs inhibitors, and SKP2 inhibitors, are being investigated [473].PROTACs and molecular glues are also being developed for the treatment of cancer.At the same time, high-throughput screening also helps researchers screen suitable inhibitors [467].Furthermore, during tumor proliferation, the UPS may influence various oncogenic signaling cascades, concurrently dysregulating multiple pathways and thereby complicating the development of targeted therapeutic strategies.Therefore, multitarget combination therapy is a direction for future development.Finally, further exploration of UPS function and clinical studies will provide important implications for the development of new cancer treatment strategies.are bifunctional small molecules consisting of two specific ligands: one for an E3 ubiquitin ligase and another that binds to a target protein.These ligands are connected by a linker, creating a trimeric complex-target protein ligand-Linker-E3 ligand.The E3 ligase tags the target protein with ubiquitin, leading to its specific degradation through the ubiquitin-proteasome pathway.Molecular glue is used in the field of drug discovery and biochemistry to refer to a small molecule that promotes the interaction between two proteins, typically leading to a therapeutic effect.These molecules act by "gluing" a target protein and a ubiquitin ligase together, facilitating the ubiquitination and subsequent degradation of the target protein by the proteasome system.
for Returned Scholar, Zhengzhou University (grant number: 32213234, F. Liu), the Natural Science Foundation of Henan province (grant number: 242300421308, X. Chen), the Postdoctoral Fellowship Program of CPSF (grant number: GZC20230741, X. Jia), and the Central Plains Science and Technology Innovation Leading Talents (grant number: 224200510015, K. Liu).

Fig. 3
Fig. 3 Ubiquitination and deubiquitination regulation of the hallmarks of cancer.E3 ubiquitin ligases and deubiquitinating enzymes, by regulating the degradation and stability of proteins, significantly influence the hallmarks of malignant tumors, which include sustained proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, genome instability and mutation, tumor-promoting inflammation, reprogramming energy metabolism, evading immune destruction, unlocking phenotypic plasticity, nonmutational epigenetic reprogramming, polymorphic microbiomes, and senescent cells.Each cancer hallmark-associated E3 ligase and deubiquitinating enzyme (DUB) is listed in the corresponding box

Table 1
E2 enzymes, E3 ligases, and DUBs in the regulation process of cancer hallmarks

Table 2
Summary of pharmacological strategies direct targeting the ubiquitin proteasome system for cancer therapy in clinical trials (information was obtained from https:// www.clini caltr ials.gov/)

Table 3
Representative small molecules targeting protein degradation under clinical evaluation (information was obtained from https:// www.clini caltr ials.gov/) HOIL-1, and SHARPIN that regulates signaling pathways and cellular processes by adding linear ubiquitin chains to specific target proteins.In cancer, its substrates include NEMO, CYLD, TNFR1, RIPK1, and MLKL, which play crucial roles in the NF-κB signaling pathway.